Preferential association of nucleolar organizing human chromosomes as revealed by silver staining technique at mitosis

Summary The frequency of different types of satellite associations of nucleolar organizing human chromosomes (i.e. acrocentric chromosomes; 13, 14, 15, 21, and 22) is reported using 10 normal individuals by Ag-staining technique. The preferential involvement of acrocentric chromosomes in satellite association is suggested. Only acrocentric chromosomes with active NORs (i.e. Ag-stained) were found in association while unstained (inactive NORs) chromosomes were never seen in satellite association. In general as number of NORs expression increase, the frequency of association per cell was also increased. A possible mechanism and the clinical consequences of such an unusual phenophenon is described.     

The relationship between the frequency of participation of acrocentric chromosomes in formation of satellite associations and the duration of human lymphocyte cultivation with phytohemmaglutinin]

All acrocentrics were shown to take part in the formation of satellite association at the 52nd, 72nd and 90th hr of human lymphocytes culturing, in the first mitosis with equal frequency but having different associative capacity in further mitoses. It seems likely that a single nucleolus in the lymphocytes at initial stage of activation, the high frequency of satellite associations, and a random participation of acrocentrics in the associations in first mitosis are due to that all acrocentrics form association in small lymphocytes which are sensitive to PHA.     

Cytogenetic damage in plastic industry workers exposed to polyvinyl chloride

Cytogenetic investigations on peripheral blood lymphocytes of 40 workers exposed to polyvinyl chloride in the plastic industry were undertaken. These were compared with an equal number of occupationally unexposed and matched controls in relation to age, sex and smoking habits. The mitotic index (MI), chromosomal aberrations (CA), sister chromatid exchanges (SCE) and satellite associations (SA) were analysed. All the parameters showed a significant increase (p <0.01) in the exposed sample compared with the controls, viz MI, 3.64-6.30, CA 1.02-3.77, SCE 3.40-7.83 and SA 5.57-12.05. The occurrence of the DG type of satellite association B was highest and that of 3D type lowest. The frequencies of all the parameters increased with the duration of exposure, but MI declined after 15 years of exposure.     

Differences between cystic fibrosis and normal cells in the degree of satellite association

Summary The degree of satellite association was found to be significantly higher in phytohemagglutinin (PHA)-stimulated lymphocytes from cystic fibrosis (CF) patients than from those of control individuals. PHA-stimulated lymphocytes from obligatory heterozygotes for the CF mutant allele showed an intermediate degree of satellite association. The degree of satellite association was estimated by the frequency of cells exhibiting associations, by the number of associations per cell, and by the number of chromosomes in an association. The differences in the degree of satellite association were dependent on the concentration of colchicine used for cell arrest. These findings may assist in developing a diagnostic method for the early identification of heterozygotes for the CF allele and for prenatal detection of CF homozygous fetuses.This paper is based on a portion of a dissertation to be submitted by Y. Ravia in partial fulfilment of the Ph. D. requirements in the Graduate School of Tel Aviv University     

Intercellular NOR-Ag-variability in man. II. Search for determining factors,clonal analysis

Summary Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts.In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

Genetic determination of NOR activity in human lymphocytes from twins

Summary Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Agstained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag+NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acrocentric association frequency were not significantly higher in DZ compared with MZ twins.     

Alignment of BrdU-substituted chromatids in satellite association in human metaphase cells

We have studied the orientation of BrdU-substituted chromatids in satellite associations in cells double-stained to reveal both the Ag/As nucleolar organizer regions and, simultaneously, sister chromatid differentiation. In those satellite associations with all four chromatids joined by Ag stain, and with the axes in a virtually straight line, we have observed an excess of concordant configurations. Where an association was considered single, dark chromatids were involved in significantly more associations than light chromatids. Within this group, the observed excess of concordant associations was not greatly different from the numbers observed in the straight, double-chromatid group of configurations. Whether the increased involvement of dark chromatids in satellite associations provides a complete explanation for the observed excess of concordant associations, or whether certain individuals show a specific tendency to form concordant associations, must await further data.     

Satellite 2 demethylation induced by 5-azacytidine is associated with missegregation of chromosomes 1 and 16 in human somatic cells

Satellite sequences are an important part of the pericentromeric regions in mammalian genomes; they play a relevant role in chromosome stability and DNA hypomethylation of these sequences has been reported in ICF syndrome and in some cancers that are closely associated with chromosomal abnormalities. Epigenetic modifications of satellite sequences and their consequences have not been extensively studied in human cells. In the present work, we evaluated satellite 2 methylation patterns in human lymphocytes exposed to 5-azacytidine (5-azaC) and assessed the relationship between these patterns and chromosome missegregation. Human lymphocytes were exposed to 10μM 5-azaC for 24, 48, and 72h. Segregation errors were evaluated in binucleate cells using FISH against pericentromeric regions of chromosomes 1, 9, and 16. DNA methylation patterns were evaluated by immunodetection, and by bisulfite plus urea conversion and sequencing. We have identified that 5-azaC induced missegregation of chromosomes 1 and 16, which have highly methylated satellite 2, after 72h of exposure. Chromosome methylation patterns showed a notable decrease in pericentromeric methylation. Bisulfite conversion and sequencing analysis demonstrated demethylation of satellite 2 associated to 5-azaC exposure, principally after 72h of treatment. This change occurred in a non-specific pattern. Our study demonstrates an association between loss of satellite 2 DNA methylation and chromosome loss in human lymphocytes.     

Support for random alignment of mitotic chromatids in associating nucleolus organizers

Summary Peripheral blood cultures of five healthy chromosomally normal adults were used to study the lateral orientation of mitotic chromatids in satellite associations. Chromosomes were prepared after bromodeoxyuridine substitution for two S phases and the fluorescence-plus-Giemsa (FPG) technique. Conventionally stained preparations were used to assess the polymorphisms of the acrocentrics in each individual. Satellite association pairs in which the acrocentrics were involved in a close, relatively straight end-to-end configuration were analyzed in cells with differentially stained sister chromatids. The number of concordant (light-light) and discordant (light-dark) chromatid alignments in associations varied from individual to individual. Chi square analysis revealed that four of the five subjects and the combined cell population from all subjects showed no deviation from the expected frequency of random alignment. The one subject with preferential nonrandom alignment had the widest range of polymorphisms and very long stalks involved in the majority of the associations, compared with the rest. We have obtained no evidence that as a general rule satellite associations are nonrandom with preferential orientation of dark-to-dark and light-to-light chromatids, although this may be the case in some individuals with very active NORs.     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.     

N-Band polymorphism of human acrocentric chromosomes and its relevance to satellite association

Summary With the aid of Q- and N-banding techniques we investigated the relationship between the length of satellite stalks, the appearance of N-bands and the frequency of satellite association of individual acrocentric chromosomes in the cells of seven individuals, including one male with a satellited and small Y-chromosome. The appearance of N-bands seemed to be a constant and characteristic property of individual acrocentric chromosomes, independent of the status of concentration of the chromosomes at metaphase. The homolog with longer satellite stalks had larger N-bands and participated in satellite association at a higher frequency than the one with shorter stalks. It appeared that N-bands were present along the whole length of the satellite stalk, the size of which could possibly reflect the amount of rDNA present in the nucleolar organizers in human chromosomes.     

A Highlights from MBoC Selection: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.     

Infertility associated with two accessory bisatellited chromosomes

Summary Two extra bisatellited chromosome identified as inv dup (15) (pterq11.2::q11.2pter) were found in an oligoasthenospermic male. Analysis of Ag-staining in the proband and in one fertile brother with a normal karyotype revealed that nucleolar organizer region (NOR) activity was significantly increased in the patient. The frequency of satellite associations was also significantly higher in the index case, but no correlation was found between NOR activity and acrocentric associations. These results suggest that extra NOR activity and the elevated frequency of satellite associations could predispose to gametogenic impairment.     

Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.     

Human nucleolar organizer chromosomes: satellite associations

The D and G group chromosomes from cultured human lymphocytes exhibit single and multiple satellite associations when stained with silver. Unlike earlier methods this simple and highly repeatable procedure shows physical attachments between satellited regions of various acrocentric autosomes. After studying 1,000 satellite associations from 118 normal individuals, it was found that both single and multiple associations occur with frequencies that correlate with random expectancies.     

The origin of mitotic sex-chromosome association in the brush-tailed possum, Trichosurus vulpecula (marsupalia:phalangeridae)

Nonrandom associations between the sex chromosomes of the brush-tailed possum, Trichosurus vulpecula, were found to be due to association of nucleolar organizer regions (NOR's) on the X and Y chromosomes. NOR association was also observed between an autosome and the X chromosome. These findings, based on silver staining, are in contrast to the report of MURRAY (1977), who observed sex-chromosome association in this animal and indicated that these nonrandom associations may reflect an association between heterochromatic regions during interphase. We observed only two pairs of NOR's per cell in this animal, one autosomal and one on the sex chromosomes, rather than the several such regions observed by MURRAY, who used an N-banding technique. We discuss the problem of nonhomologous chromosome association in mammalian cells as influenced by heterochromatin and NOR's and find little support for nonhomologous chromosome associations at mitotic metaphase due to heterochromatin association.     

BUdR-Giemsa labeling and satellite association in human leukocytes

Summary Satellite associations were analysed in differentially stained human leukocyte chromosomes, obtained from four patients with Down's syndrome and four normal probands. A particular type of close association between two acrocentrics, showing a non-random arrangement of sister chromatids in a concordant dark-to-dark and light-to-light alignment, was found to be more common in patients with Down's syndrome compared with the normal controls. Apart from this particular type of association, sister chromatids are randomly arranged in satellite associations between two acrocentrics in both groups of probands. Considerable differences in the mean frequencies of satellite associations between first and second metaphases of the same individual were found in some probands of both groups of individuals. Since a high degree of inter-individual variability in the proliferative response of human leukocytes in culture is well established, the use of BUdR-Giemsa labeling for comparative analysis of satellite association frequencies is suggested.